SUMMARY Overall treatment failure for cutaneous leishmaniasis (CL) in Latin America is estimated at 24%. The relationship between drug susceptibility of Leishmania and therapeutic response is uncertain for all forms of human leishmaniasis. Methodological disparities and the multifactorial basis of the outcome of treatment confound attempts to correlate drug susceptibility with clinical response and impede the use of drug susceptibility in treatment policy or decisions for control and management of leishmaniasis. Comprehensive analysis of bacterial and fungal susceptibility to diverse antimicrobial drugs and the predictive utility of susceptibility testing for therapeutic outcome has shown that host factors influence the correlation with outcome of treatment. Nevertheless, overall experience in the analyses of antimicrobial drug susceptibility has consistently shown that therapeutic response is significantly lower among individuals infected with resistant organisms in a relation referred to as the ?90-60 rule?. We will apply the lessons and principles of antibacterial and antifungal susceptibility assessment, to determine the relationship of parasite susceptibility to anti- leishmanial drugs with the clinical and parasitological response to standard-of-care treatment and identify parasite factors defining this relationship. To achieve this goal three specific aims will be pursued: 1. Define the relationship between in vitro and ex vivo drug susceptibility phenotype of clinical strains and the clinical and parasitological response to treatment in CL patients, 2. Determine the reciprocal effect of drug susceptibility phenotype on activation of innate immune/inflammatory responses and its relationship with outcome of treatment, and 3. Identify genetic markers of Leishmania associated with drug susceptibility phenotype and therapeutic outcome We will strategically exploit an extensive collection of Leishmania strains and corresponding clinical data base available from patients who have participated in previously concluded randomized clinical trials, and patients prospectively identified in Project 1 through active case detection. Susceptibility of these clinical strains to pharmacologically relevant concentrations of meglumine antimoniate and miltefosine will be determined in novel in vitro and ex vivo analytical systems that approximate the in vivo cellular environment and confounders of clinical response will be controlled in evaluating the relationship with treatment outcome. Genetic markers and underlying mechanisms of therapeutic outcome will be identified by SNP profiling from genome-wide transcriptomes of drug-susceptible and resistant Leishmania from therapeutically responsive and non- responsive CL. Results of this project will establish the contribution of drug resistance in treatment failure, guide optimization of current treatment, development of more effective treatments, and inform treatment policy.